Frequency modulation of a bacterial quorum sensing response

In quorum sensing, bacteria secrete or release small molecules into the environment that, once they reach a certain threshold, trigger a behavioural change in the population. As the concentration of these so-called autoinducers is supposed to reflect population density, they were originally assumed to be continuously produced by all cells in a population. However, here we show that in the α-proteobacterium Sinorhizobium meliloti expression of the autoinducer synthase gene is realized in asynchronous stochastic pulses that result from scarcity and, presumably, low binding affinity of the key activator. Physiological cues modulate pulse frequency, and pulse frequency in turn modulates the velocity with which autoinducer levels in the environment reach the threshold to trigger the quorum sensing response. We therefore propose that frequency-modulated pulsing in S. meliloti represents the molecular mechanism for a collective decision-making process in which each cell’s physiological state and need for behavioural adaptation is encoded in the pulse frequency with which it expresses the autoinducer synthase gene; the pulse frequencies of all members of the population are then integrated in the common pool of autoinducers, and only once this vote crosses the threshold, the response behaviour is initiated.


Supplementary Information
Frequency modulation of a bacterial quorum sensing response Vera Figure 1. SinR has a degenerated AHL binding site. a Amino acid sequence alignment of the AHL binding domains of 10 LuxR-type regulators illustrating changes of SinR in residues crucial for AHL coordination. SmeSinR, Sinorhizobium meliloti SinR (Uniprot accession number Q92PD1); SmeExpR, S. meliloti ExpR (Q2HY11); AtuTraR, Agrobacterium tumefaciens TraR (P33905); SfrTraR, Sinorhizobium fredii NGR234 TraR (P55407); CviCviR, Chromobacterium violaceum CviR (D3W065); AfiLuxR, A. fischeri LuxR (P12746); PaeLasR, Pseudomonas aeruginosa LasR (P25084); PaeRhlR, P. aeruginosa RhlR (P54292); PaeQscR, P. aeruginosa QscR (Q9RMS5); EcoSdiA, Escherichia coli SdiA (P07026). Arrows mark residues involved in coordination of the lactone ring and the 3-oxo moiety of the AHL in the crystal structure of A. tumefaciens TraR 1 (see b), numbers above indicate positions in TraR (black) and SinR (turquois) amino acid sequences. Numbers on the left and right denote the first and last residues shown in the alignment. Residues with 60% or higher conservation are indicated by boxes according to the Clustal X Colour Scheme 2 . b 3D structure of A. tumefaciens TraR (PDB entry 1L3L) 1 (left). The AHL is coordinated by hydrogen bond interactions provided by tyrosine (Y53), tryptophan (W57) and aspartate (D70). Threonine (T129) coordinates a water molecule which in turn establishes a hydrogen bond to the 3-oxo moiety of the AHL. The SinR AHL binding site (right) is modelled based on A. tumefaciens TraR (PDB entry 1L3L). Three of the four residues coordinating the AHL in TraR are exchanged in SinR for residues lacking the potential to establish hydrogen bonds with an AHL molecule, providing a structural basis for SinR being irresponsive to AHLs 3 .

Supplementary Figure 2. Stochasticity in sinI expression. a
Phase contrast and fluorescence microscopy snapshots (top) of wild-type (wt) and expRcolonies carrying two copies of the sinI promoter fused to mCerulean and mCherry fluorophore genes, respectively, representing exemplary raw data for Fig. 1b. In total, 10 (wt) and 12 (expR -) colonies were imaged on 3 different days, all with similar results. Scale bars, 2 µm. Simplified sketch (bottom) of the respective promoter-fluorophore gene fusions. Both the sinI promoter-mCerulean and the sinI promoter-mCherry fusion are located on the same suicide plasmid integrated into the S. meliloti chromosome at the native sinI locus. The two promotor-fluorophore fusions are separated by approx. 4 kb of plasmid DNA. The plasmid also comprises a constitutive promoter-mVenus fusion for segmentation of microscopy images; the strains furthermore possess a third, dark copy of the sinI promoter regulating expression of sinI. b Flow cytometry analysis (top) of wt and expRcells likewise carrying two sinI promoter-fluorophore gene fusions during exponential growth confirm strong stochasticity of sinI expression. Each plot indicates fluorescence intensities of 15,000 cells measured in one experiment. Blue, cells carrying the fluorophore gene fusions; grey, respective background controls. Rectangles indicate the 'negative' gate comprising at least 99.9% of background controls. Data is representative of 3 independent experiments. Simplified sketch (bottom) of the constructs employed for flow cytometry: A sinI promoter-mScarlet-I fusion is integrated into the chromosome in between the native sinI promoter and the sinI coding sequence (i.e., at the native sinI locus) by double homologous recombination, and a sinI promoter-mNeonGreen fusion is located on a suicide plasmid integrated into the single-copy megaplasmid pSymB in the intergenic region between the exoP and the thiD gene; pSymB is essential for S. meliloti viability unless crucial elements are first transferred onto the chromosome 4 . The control strains lack the fluorophore on the chromosome and carry a promoterless mNeonGreen on an otherwise identical suicide plasmid likewise integrated in the exoP-thiD intergenic region. Figure 3. sinI expression rate. a Plots of mean fluorescence intensities from the PsinI-mVenus fusion (top), the time derivatives of these mean fluorescence intensities (middle), and the corresponding sinI expression rates (bottom) of the expRmicrocolony from Fig. 1c over the whole experiment run time (first column) and three more expRmicrocolonies (columns two to four). Once a pulse has ended, no more fluorophores are produced, and mean fluorescence intensities slowly decline due to dilution by cell growth and/or degradation. Since the time derivatives represent the change in mean fluorescence intensities over time, they decrease as soon as fluorophore production has passed its maximum; they become negative once it completely petered out, as they then only represent the effects of fluorophore dilution and/or degradation. To deduce the actual expression rate, the time derivative is set off against a term for the dilution of fluorophores by cell growth, and a term for their -albeit small -degradation rate. In principle, the expression rate should be either 0 or above, but small negative values are possible due to inaccuracies, e.g., in single fluorescence measurements, determination of cell elongation rates (feeding into the term for fluorophore dilution), or fluorophore degradation rate. Both the time derivative and the expression rate represent fits to the data calculated over a sliding window of 11 time points; see Methods for details. In every plot, the maxima of two sinI expression pulses -i.e., the measurements ensuing the highest fluorophore production rate -are marked with broken lines. b Compilation of pulse frequencies per cell life time, and per cell life time and hour from 9 expRcolonies imaged on 3 different days. Respective means are given and indicated by the bars, error bars indicate standard deviations, open circles indicate individual data. N = 2,517 cells. Figure 4. SinR is very unstable and scarce. a Western blots of cell lysates identifying the Flag-tagged SinR (F-SinR) band (left) and indicating rapid F-SinR degradation after chloramphenicol treatment at 1-minute intervals (right). Numbers on the left indicate molecular weight standards. Protein stability assay representative of 3 biological replicates, see (b) for analysis. b Plots of relative abundance of F-SinR and DnaK (left) and F-SinR normalized to DnaK (right), each fitted with a model of one phase exponential decay. The fits were constrained with K > 0 (i.e., a degradation over time) and plateau = 0 and yield a half-life of 3.1 minutes for F-SinR [Y(0) = 1.12, R 2 = 0.87] and of 4.0 minutes for F-SinR/DnaK [Y(0) = 1.08, R 2 = 0.78]. Data for F-SinR alone is given in the main text and Fig. 2b since standard deviation in DnaK data -and thus F-SinR/DnaK data -is larger than for F-SinR data alone. Degradation of DnaK could not be fitted. Data, means ± standard deviations of 3 biological replicates. c Fluorescence microscopy images of cells of an expRcontrol strain (left) and an expRstrain carrying the mScarlet-I-sinR fusion (right). White square, area displayed in Fig. 2c (left). Scale bars, 2 µm. Images representative of 1,670 (expR -) and 2,293 (mScarlet-I-sinR expR -) cells, respectively, imaged on 3 different days (see Fig. 2c (right) for quantification). d Histograms of fluorescence intensities from the sinI promotor-mVenus fusion either with native sinR (light blue), or flag-sinR or mScarlet-I-sinR (dark blue), expressed from the native sinR promoter suggest that native, i.e., untagged SinR is even less stable and/or less abundant than suggested by western blot analysis and single molecule microscopy. Each histogram indicates fluorescence intensities of 15,000 cells measured in one experiment and is representative of 3 biological replicates. Total number of cells analysed: N = 45,000 per strain. Figure 5. SinR as a key factor. a Trajectories showing sinI expression pulses in colonies with different levels of sinR transcription, related to Fig. 2d. b Representative flow cytometry data (left) confirming relative differences in sinI expression pulse frequency: Histograms of fluorescence intensities from 15,000 cells carrying the sinI promoter-mVenus fusion (blue) with the same genetic modifications as in (a) and Fig. 2d, and 15,000 cells of the respective control strains without a fluorophore gene fusion (grey); the latter were used as donor strains for the experiment in Fig. 4b. Bar plots (right) indicate the fraction of cells in the samples displaying fluorescence intensities higher than those of the control strains in 3 biological replicates and their means and standard deviations. Statistical test, Welch's ANOVA test with post-hoc Dunnett's T3 multiple comparisons test. **, P < 0.01; ***, P < 0.001. Multiplicity-adjusted P values: PsinR* vs. native 0.0012, PsinR* vs. nurR ++ 0.0010, native vs nurR ++ 0.0015. Total number of cells analysed: N = 45,000 per strain. c Representative flow cytometry data (left) verifying that nurR exerts its effect on sinI expression via increasing SinR levels and not directly: nurR overexpression has no effect on fluorescence intensities from the sinI promoter-mVenus fusion in the sinRstrain, and hardly any on the strain carrying the sinR promoter mutation. Histograms of 15,000 cells carrying the sinI promoter-mVenus fusion (blue) and 15,000 cells of the respective control strains (grey) per genetic background. Bar plots (right) indicate the fraction of cells in the samples displaying fluorescence intensities higher than those of the control strains in 3 biological replicates and their means and standard deviations. Total number of cells analysed: N = 45,000 per strain. d Representative fluorescence microscopy image (left) and flow cytometry data (right) illustrating that direct overexpression of (mScarlet-I-)sinR disrupts the otherwise stochastic regulatory system: It not only abolishes heterogeneity in fluorescence, but also greatly augments fluorescence intensities. Fluorescence image acquired with identical settings as in Supplementary Fig. 4c, upper part reproduced with identical dynamic range as in Supplementary Fig. 4c, lower part with a 10fold wider dynamic range. Scale bar, 2 µm. Flow cytometry data acquired with identical settings as in (b); blue, 15,000 cells carrying the sinI promoter-mVenus fusion; grey, 15,000 cells of the respective control strain. Total number of cells analysed: N = 589 for SMM data, 45,000 per strain for flow cytometry data, all in 3 biological replicates. Figure 6. Quantification of pulse data is robust to changes in peak prominence threshold. To test whether our results might have been falsified by our choice of threshold for what is considered a pulse, the data set with different sinR expression levels ( Bar plots (middle) show mean PsinI-mVenus expression pulse frequencies for 9 colonies imaged on 3 different days determined with the respective threshold, and means and standard deviations of each data set; different thresholds yield different absolute pulse frequencies, but do not change relative differences between different genetic backgrounds. Statistical tests, Welch's ANOVA tests with post-hoc Dunnett's T3 multiple comparisons test. ns, not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Multiplicity-adjusted P values: PsinR* vs. native 0.0181, PsinR* vs. nurR ++ < 0.0001, native vs nurR ++ 0.0003 for prominence threshold 3 (a), PsinR* vs. native 0.0290, PsinR* vs. nurR ++ < 0.0001, native vs nurR ++ < 0.0001 for prominence threshold 6 (b), PsinR* vs. native 0.0797, PsinR* vs. nurR ++ < 0.0001, native vs nurR ++ < 0.0001 for prominence threshold 12 (c). Bar plots (right) indicate the fraction of cells in the samples displaying fluorescence intensities higher than those of the control strains in 3 biological replicates and their means and standard deviations. Statistical tests, two-tailed unpaired t-tests with Welch's correction for (b, f); Welch's ANOVA test with post-hoc Dunnett's T3 multiple comparisons test for (d). **, P < 0.01. P values (multiplicity-adjusted, if appropriate): rich vs. P-starv in expR -0.0059 (b); expRvs expR -dgc0 0.0012, expRvs. expR -pde0 0.0079, expR -dgc0 vs. expR -pde0 0.0032 (d); expRvs. wt, 0.0021 (f). g Bar plots of single molecule microscopy data indicate that expR does not affect mScarlet-I-SinR levels. Open circles, data from 3 biological replicates; bars, means and standard deviations. Statistical test, two-tailed unpaired t-test with Welch's correction. ns, not significant; P = 0.1408. N = 1,300 (expR -), 1,503 (wt). h Histograms of fluorescence intensities from the PsinI-mVenus fusion in a sinR -expRstrain (light blue) and the same strain overproducing His-GB1-SinR (dark blue) indicate in vivo activity of the fusion protein. Data representative of 2 biological replicates. i Western blot of Flag-tagged SinR representative of 2 biological replicates (left) and quantification of relative protein abundances in both replicates (right) confirm relative differences in SinR abundance under rich growth conditions vs. phosphate starvation in expRbackground and expR -, expR -dgc0 and expR -pde0 strains determined by single molecule microscopy; likewise, western blot data confirms that presence of expR in the wild-type background does not increase of F-SinR levels. Figure 8. The expR -pde0 strain displays elevated c-di-GMP levels. a, b, c Bar graphs indicating elevated c-di-GMP levels (a), increased attachment detected by crystal violet staining (b), and decreased motility on soft agar plates (c) of the expR -pde0 strain compared to the expRparental strain. Bars represent means, error bars represent standard deviations, and open circles represent data from 3, 6 and 5 underlying biological replicates, respectively. Statistical tests, two-tailed unpaired t-tests with Welch's correction. **, P < 0.01; ****, P < 0.0001. a Whereas 2.9 ± 0.27 pmol c-di-GMP per mg protein were detected in exponential phase samples of the expRparental strain, and 0.5 ± 0.21 pmol c-di-GMP per mg protein in expRstationary phase samples, 124.8 ± 7.27 pmol c-di-GMP per mg protein were detected in exponential phase samples of the expR -pde0 strain, and 22.1 ± 2.93 pmol c-di-GMP per mg protein in expR -pde0 stationary phase samples. OD600 » 0.45, P = 0.0012 for exponential phase; OD600 > 2, P = 0.0053 for stationary phase. b Surface attachment in the expR -pde0 strain is elevated approximately 4fold compared to the parental strain (P < 0.0001), consistent with prior findings that c-di-GMP stimulates production of, e.g., arabinose-containing polysaccharide and other polysaccharides important for attachment in rhizobia [5][6][7] . Of note, these attachment-related polysaccharides are regulated differently than the exopolysaccharide galactoglucan which plays an important role in S. meliloti colony expansion and sliding motility [8][9][10] and is part of the organism's quorum sensing response 3,11 . c Motility of the expR -pde0 strain is reduced by approximately 30% compared to the parental strain (P < 0.0001), consistent with prior findings that elevated c-di-GMP levels repress swimming motility in S. meliloti 5,6 . d Measurements of optical densities every 30 minutes for 25 hours indicate no difference in growth of the expR -pde0 strain compared to the expRparental strain. Data, means ± standard deviations of 3 biological replicates. For details on construction and characterization of the expR -pde0 strain see Supplementary Methods 2. Figure 9. Potential positional and temporal effects. a, b Analysis of two data sets, each consisting of 9 colonies per strain imaged on 3 different days, with respect to potential positional effects on sinI expression pulse frequency. Cells were grouped in three bins according to their distance from the colony edge: the colony boundary (d = 0 µm), followed by a ring of intermediate distance (e.g., 0 < d < 1.6 µm), and the colony centre (e.g., d > 1.6 µm); the boundary between the last two bins was chosen to yield equal-sized groups. The boxes in the plots indicate median, 25 th and 75 th percentile of mean mVenus fluorescence intensities of individual cells from the PsinI-mVenus fusion (log-transformed to account for high skew in distribution), the whiskers indicate 2.5 th and 97.5 th percentiles, individual points indicate outliers. Comparison of the three positional subgroups for each strain yielded small but significant effects for (a) the data set with different sinR expression levels ( Fig. 2d) 3.2875 for wt). Mean fluorescence intensities were used as a proxy for pulses since pulse frequency must be determined over time, but cell position within the colony is bound to change over time. Statistical tests, Kruskal-Wallis tests; ns, not significant; **, P < 0.01; ****, P < 0.0001. Exact P values: < 0.0001 (PsinR*), 0.0010 (native), < 0.0001 (nurR ++ ) at ~100 cells; < 0.0001 (PsinR*), 0.0078 (native), < 0.0001 (nurR ++ ) at ~50 cells; 0.8698 (expR -), 0.3275 (wt) at ~50 cells. c, d Similarly, developing microcolonies were analysed separately for the first, second and third fourhour period of the experiment. Plots show pulse frequencies for 9 colonies per strain and respective means and standard deviations for each observation period. Comparison of the temporal subgroups for each strain did not yield significant differences neither for (c) the data set with different sinR expression levels, nor for (d) the data set comparing receptor mutant (expR -) and wild type. Statistical tests, Welch's ANOVA tests; ns, not significant. Figure 10. mScarlet-I-SinR spots are homogeneous. Single molecule movies had been recorded with the aim to establish the frequency of mScarlet-I-SinR spots in S. meliloti populations; nevertheless, we can draw some conclusions on the nature of the observed spots with respect to the number of fluorophores they contain. a Histogram of fluorescence intensities of all spots detected in mScarlet-I-sinR strains in this work (Fig. 2c, 3a, b, Supplementary Fig. 7g) (N = 3,277) except for the mScarlet-I-sinR overexpression strain ( Supplementary Fig. 5d). The frequency distribution of spot intensities can be fitted with a log-normal distribution (orange line), indicating a single, homogeneous population. Fitting models with multiple populations reduce to a single population and do not take the small increase between 70-100 photons into account that might be indicative of a second population. Taken together, this suggests that the mScarlet-I-SinR spots represent a largely homogeneous SinR population rather than a mixture of higher-order multimers 12 . Furthermore, the low intensities per spot, i.e., the very low number of emitted photons, indicates that the fluorescent spots are low copy number mScarlet-I-SinR spots, i.e. either SinR monomers or dimers. b Representative fluorescence intensity traces for selected spots, showing clear bleaching events of single fluorophores. The large majority of traces with bleaching events only have a single bleaching event until the background level is reached (left), but we also find some traces with two bleaching events (right). The step heights of bleaching events are the same for all traces (~ 70-90 AD counts). In addition, short fluorescence fluctuations are present in all traces, caused by brief ON-and OFF-blinking events of single mScarlet-I fluorophores. Taken together, this suggests a large population of mScarlet-I-SinR monomers and a small population of dimeric spots. Because our samples were chemically fixed, we cannot distinguish which of them are target-bound and which of them are part of a free, cytosolic population. Importantly, however, we find no evidence of higher-order multimers in both spot intensity and photobleaching trace analyses. Thus, this data adds strong evidence to the central finding of this work that heterogeneity in sinI expression is not caused by different expression levels of sinR but by the presence or absence of individual SinR molecules in each cell. Pulse amplitudes all vary 20-fold or more within strains or growth conditions. However, median pulse amplitudes do either not vary according to pulse frequencies at all (a, e), or only to a much smaller extent (c, g, i, k) (see Fig. 3f). Pulse amplitude data in (a, c, e, g, i, k) stems from strains/conditions with very different pulse frequencies, dot plots thus show varying numbers of data points and are also plotted as cumulative frequency distributions (right panels, respectively) for easier comparison. The increase in flow cytometry intensities correlating with very high pulse frequencies/fluorescing fractions are not reflected in pulse amplitude data and probably result from consecutive pulses that are still separated by time-lapse analysis, but add up in terms of total fluorescence intensities. Plots in (b, d, f, h, j, l) each show fluorescence data of 4,500 cells assessed as 'positive'. Figure 12. Effects on the quorum sensing response. a Fluorescence microscopy snapshots of microcolonies of a wild-type strain carrying the mCerulean fluorophore gene fused to a promoter driving expression of genes involved in exopolysaccharide production (PwgeA), representing exemplary raw data underlying Fig. 4a (left). Microcolonies were grown for 24 hours under rich or phosphate starvation conditions, respectively. Scale bars, 10 µm. b Means and standard deviations of mean fluorescence intensities per colony from the PwgeA-mCerulean fusion of the same 9 wild-type colonies as shown in Fig. 4a, here plotted over colony area. c Means and standard deviations of mean PwgeA-mCerulean intensities per colony of 9 wildtype and expRmicrocolonies, respectively, both grown under phosphate starvation conditions, plotted (left) over time and (right) colony area, respectively, illustrate that phosphate starvation induces expression of exopolysaccharide genes in S. meliloti even in absence of the AHL receptor, albeit with lower speed and intensity. This effect is due to additional direct regulation of the wge operon by phosphate starvation 13 . d Fluorescence microscopy snapshots of wt, dgc0 and pde0 microcolonies, all carrying the PwgeA-mCerulean fusion, representing exemplary raw data underlying Fig. 4a (right), and e means and standard deviations of mean PwgeA-mCerulean intensities per colony for 6 of the 9 colonies (rep. 1&2) shown in Fig. 4a plotted over colony area illustrate the onset of the quorum sensing response at different colony sizes depending on c-di-GMP levels. Scale bars, 10 µm. f Means and standard deviations of mean PwgeA-mCerulean intensities per colony for the third biological replicate for the c-di-GMP effect, plotted (left) over time and (right) colony area show similar relative, but different absolute behaviour. g Growth curves of the AHL indicator strain during incubation with supernatants from different strains and optical densities for Fig. 4b, controlling for possible effects of growth differences of the indicator strain on activation of the sinI promoter. The arrow indicates the time of the fluorescence measurement shown in Fig. 4b. h Growth curves of the supernatant donor strains for Fig. 4b indicate no effect on growth, i.e., no metabolic burden, by different sinR expression levels; donor strains did not carry the PsinI-mVenus fusion, i.e., expressed no fluorophore gene and are identical to the control strains for flow cytometry measurements shown in Supplementary Fig. 5b. Data represent means ± standard deviations of 2 biological replicates. Figure 13. Gating and quantification of flow cytometry data. a Gating was first performed on forward and side scatters (FSC and SSC, respectively) to remove dead cells and debris (SSC-A over FSC-A) and to exclude doublets (SSC-W over SSC-H). Subsequently, the number of samples was reduced to 15,000 events using the FlowJo Exchange DownSample plugin to ensure equal sample size. Strains lacking the sinI promoter-fluorophore gene fusion(s) with otherwise identical genetic backgrounds served as negative controls. b Cells in the read-out samples with higher fluorescence intensities than those of the respective control cells were assessed as 'positive'. The fraction of cells per sample assessed as 'positive' and their corresponding median fluorescence values were determined with FlowJo. The data shown here were derived from strains with different sinR expression levels, from left to right: the sinR deletion, the sinR promoter mutant, the native sinR promoter, the native sinR promoter while overproducing its transcription activator NurR, and direct overproduction of SinR from a plasmid. The scatter plots shown in the second, third, and fourth panel thus correspond to the blue histograms in Supplementary Fig. 5b, the data in the first panel to the blue histogram in Supplementary Fig. 5c (left), and the last panel to the blue histogram in Supplementary Fig. 5d. PsinR+27-Sal-r catgtcgacATTGAGGACAGCCTGTTGATTAG mScarlet-I-Sal-f catgtcgacATGGTTTCTAAAGGCGAAGCC mScarlet-I-TGA-B-r catggatccagatccacctgcCTTGTACAATTCATCCATACCA sinR-X-B-f cattctagaggatccATGGCTAATCAACAGGCTGTCCT sinR-E-r catgaattcTCAGATGGTGGGGATCAGAG PsinR-720-Eh-f gacatgattacgaattcGCGACCTTCTTCACCGATA pK18mobsacB-PsinR*-sinR PsinR*-r tgcaataaagcttggcaGGTGCAGTAATCCCGCTTA PsinR*-f ccaagcttTATTGCACTAGACAAAACCGG sinR-Bh-r tcgactctagaggatccTCAGATGGTGGGGATCAGAG PsinR-720-Eh-f gacatgattacgaattcGCGACCTTCTTCACCGATA pK18mobsacB-PsinR*-mScarlet-I PsinR*-r tgcaataaagcttggcaGGTGCAGTAATCCCGCTTA PsinR*-f ccaagcttTATTGCACTAGACAAAACCGG mSca-Bh-r tcgactctagaggatccCTTGTACAATTCATCCATACCA PpstS-A-F catgacgtcTTGCGATCGTCAAGCATATC pK18mob2-PpstS-mVenus PpstS-S-r catgtcgacAGATTTCATGAATGTTCTCCC 23 PsinI- M-f  catacgcgtCAACGATTCTCGGCATATCC  PsinI-mCerulean on pK18mob3-2xPsinI  PsinI-K-r  catggtaccACCGTTTCCGTTCACTATCCT  PsinI-B-f catggatccCAACGATTCTCGGCATATCC PsinI-mCherry on pK18mob3-2xPsinI PsinI-X-r cattctagaACCGTTTCCGTTCACTATCCT Plac145-AatII-f catgacgtcTTGGCCGATTCATTAATGCAG Plac-mVenus on pK18mob3-2xPsinI Plac-rbs-S-r catgtcgaccattttttcgctccatgCCTGTGTGAAATTGTTATCCGC PsinR-B-f gatggatccCGCATATTCTGTCGCCGT pK18mob3-PsinR-mCherry mCh+-S-r ATTGCGGGAGCTCTTACTTG sinRUTR-X-f catggatccggtgttacggcatgtctaga pK18mob3-sinRUTR-mCherry mCh+-S-r ATTGCGGGAGCTCTTACTTG PwgeA-K-rev catggtaccTTCCAAAGTGGCCATCTGCTT pK18mob3-PwgeA-mCerulean PwgeA-M/Xho-fwd tagacgcgttagctcgagTTCGGGAGGACTGACCTGT Ptrp-fwd oligo1 gatccgcggccgcgaaatgagctgttgacaattaatcatcg Ptrp-mCherry on pK18mob3-PwgeA-mCerulean

Supplementary Methods 1. Details on strain constructions.
For the sinI promoter-mVenus fusion analysed by time-lapse fluorescence microscopy and flow cytometry, a 678 bp fragment containing the sinI promoter, 5' untranslated region (UTR) and first nine codons of sinI, the mVenus 28 gene including stop codon, and a 729 bp fragment containing the sinI coding sequence preceded by its native ribosome binding site were cloned into pK18mobsacB 21 in a step-wise fashion using HindIII, XbaI, KpnI and EcoRI restriction enzymes (at one step taking advantage of a HindIII site within the fragment including the sinI promoter); the final construct pK18mobsacB-PsinI-mVenus-sinI thus carried the mVenus gene flanked upstream by the sinI promoter and downstream by the sinI coding sequence. After conjugation and double homologous recombination in S. meliloti, the chromosomal sinI locus comprises the native sinI promoter, UTR and first nine codons followed by an XbaI site, the mVenus gene including a KpnI site immediately preceding the stop codon, and the sinI coding sequence preceded by its native ribosome binding site.
The sinI promoter-mScarlet-I fusion analysed by flow cytometry was constructed in a similar fashion as the sinI promoter-mVenus fusion. However, here the sinI gene is preceded by the entire sinI UTR. Fusion of the flanking regions with codon-optimized mScarlet-I 29 was performed by ligase cycling reaction 30 , and restriction digestion followed by ligation was only employed for cloning of the preassembled insert into pK18mobsacB, resulting in pK18mobsacB-PsinI-mScarlet-I-sinI. Thus, S. meliloti strains carrying this fusion at the native sinI locus have no restriction site introduced by cloning in between the sinI promoter, UTR and first nine sinI codons and the downstream fluorophore gene, and no restriction site in between the fluorophore gene and the downstream sinI UTR and coding sequence.
The sinI promoter-mNeonGreen fusion likewise analysed by flow cytometry comprises the 259 bp sequence upstream of the sinI transcription start site (TSS), again followed by the sinI UTR and first nine sinI codons, fused to codon-optimized mNeonGreen 31 via overlap extension PCR 32 . Cutting with KpnI and EcoRI restriction enzymes, the assembled insert was cloned into a suicide vector carrying a 964 bp fragment amplified from S. meliloti megaplasmid pSymB, i.e., the 3' 676 bp of the exoP gene and the subsequent 288 bp including a terminator sequence immediately downstream of exoP. Thus, in S. meliloti the final construct pKE-PsinI-mNeonGreen integrates into the essential megaplasmid pSymB 4 by homologous recombination, and readthrough of exoP transcription into the sinI promoter-mNeonGreen fusion is blocked by the included terminator sequence (as demonstrated for the analogous promoter probe vector pSRPP18 33 ). The corresponding control plasmid pKE-mNeonGreen carrying a promoterless mNeonGreen was obtained by amplification of only the codon-optimized mNeonGreen gene with the same reverse primer and an mNeonGreen forward primer including a KpnI site.
The construct comprising two identical copies of the sinI promoter studied by fluorescence microscopy is based on a modified version of a triple reporter cassette 24 allowing for parallel monitoring of the activities of up to three different promoters fused to the mCerulean 34 , mCherry 35 and mVenus genes, respectively; in contrast to the earlier version, the modified cassette includes the rpoC/thrA tandem terminator inserted between the mCerulean and mCherry genes, and only two copies instead of four of the T1 transcription terminator between the mCherry and mVenus genes 27 . Both sinI promoter-fluorophore gene fusions comprise the same 259 bp sequence upstream of the sinI TSS as the sinI promoter-mNeonGreen fusion, again followed by the sinI UTR and the first nine sinI codons; these fragments were cloned in front of mCerulean using MluI/KpnI restrictions sites and in front of mCherry using BamHI/XbaI restriction sites, respectively. The triple reporter cassette was then cut out of its original vector pK18mob2 by cutting with EcoRI/HindIII restriction enzymes (and additionally with MreI to allow for separation of fragments via gel electrophoresis) and cloned into the EcoRI/HindIII-digested suicide plasmid pK18mob3, derived from pK18mob2 21 by deletion of the lac promoter preceding the multiple cloning site. Furthermore, to allow for microscopy image segmentation, a 174 bp fragment including the lac promoter amplified from pSRKGm 23 combined with the sinI ribosome binding site was cloned in front of the mVenus gene using AatII/SalI restriction enzymes. In both S. meliloti strains carrying the final construct pK18mob3-2xPsinI in this work, the vector had integrated into the chromosome via the sinI promoter-mCherry fusion, so that the two fusions are separated by approximately 4 kb of plasmid DNA; a third, dark copy of the sinI promoter regulates expression of sinI in these strains (Fig. 1b & Supplementary Figure 2a).
The sinR promoter-mCherry construct analysed by fluorescence microscopy in turn is a derivative of the above 2xPsinI construct. The entire sinR promoter-mCherry fusion (including a 294 bp fragment of sinR promoter, UTR and start codon) was amplified from an earlier triple reporter cassette located on pK18mob2 27 and used to replace the sinI promoter-mCherry fusion by cutting both the PCR product and the double sinI promoter-fluorophore vector with BamHI/SacI. Thus, this construct pK18mob3-PsinR-mCherry likewise carries a lac promoter-mVenus fusion for microscope image segmentation, and the sinI promoter-mCerulean fusion. The corresponding control plasmid pK18mob3-sinRUTR-mCherry lacks the sinR promoter, but includes the sinR ribosome binding site to enable translation of potential transcriptional readthrough. Both in the S. meliloti strain carrying the read-out construct and the S. meliloti strain carrying the control construct the plasmid integrated into the chromosome via the sinI promoter-mCerulean fusion.
The wgeA promoter-mCerulean construct assayed in long-term time-lapse fluorescence microscopy is a derivative of yet another triple reporter cassette, carrying a 303 bp fragment including the wgeA promoter, UTR and the first five wgeA codons in front of mCerulean inserted via KpnI/MluI restriction sites 27 . The trp promoter and UTR combined with the lac ribosome binding site was assembled from oligonucleotides and inserted in front of mCherry via BamHI/XbaI restriction sites to allow for microscope image segmentation. The mVenus gene and preceding restriction sites and T1 tandem terminators in turn were removed and exchanged for a short terminator sequence in order to allow use of this construct in combination with the chromosomal sinI promoter-mVenus fusion. The final cassette comprising the wgeA promoter-mCerulean fusion, the rpoC/thrA tandem terminator and the trp promoter-mVenus fusion was cloned into pK18mob3 via EcoRI and HindIII restriction sites; in S. meliloti strains carrying this construct pK18mob3-PwgeA-mCerulean the plasmid integrated into pSymB via the wgeA promoter-mCerulean fusion.
The pstS promoter-mVenus fusion employed to verify the onset of phosphate starvation in timelapse fluorescence microscopy experiments likewise is part of a triple reporter construct, albeit on pK18mob2; a 298 bp fragment including the pstS promoter and the first three pstS codons was cloned in front of the mVenus gene using AatII and SalI restriction enzymes. The final plasmid (that also includes the above-described sinR promoter-mCherry fusion, and the above described sinI promoter-mCerulean fusion) was integrated into the S. meliloti sinI mutant Sm2B4001 20 , so that the resulting scavenger/indicator cells do not contribute to AHL levels on the agarose pad during experiments.
The AHL indicator plasmid used in the plate reader experiment was built by cutting both the original pK18mob2-triple reporter cassette 24 and pSRKGm 23 with EcoRI/HindIII, thus removing lacI, the lac promoter and part of lacZa from the latter, and subsequent ligation of the pSRKGm backbone and the triple reporter cassette, yielding pSRKGm-indicator.
For the mScarlet-sinR translational fusion employed in single molecule microscopy, a 733 bp fragment containing the sinR promoter, UTR and first nine codons, the mScarlet-I gene without stop codon, but including a linker (AGGSGS) added via a primer tail, and the sinR coding sequence were cloned into pK18mobsacB in a step-wise fashion using SalI/HindIII, EcoRI/XbaI and BamHI/SalI restriction enzymes. After conjugation and double homologous recombination in S. meliloti, the fusion protein was expressed from the native sinR promoter.
For the N-terminally 3xFLAG-tagged SinR, a 709 bp fragment containing the sinR promoter and the sinR gene were amplified separately, with the coding sequence for the 3x-FLAG tag (DYKDHDGDYKDHDIDYKDDDDK) and the linker (GSGSGS) comprised as overlapping tails in the inner primers; the two fragments were then fused via overlap extension PCR and cloned into pK18mobsacB using PstI (cutting within the sinR promoter fragment) and EcoRI. The construct was subsequently integrated into the S. meliloti genome via double homologous recombination, replacing the native sinR gene, so that strains carrying this construct produced 3xFLAG-tagged SinR from the native sinR promoter.
For production and purification of His6-GB1-SinR, the sinR gene was cloned via NcoI/BamHI restriction sites into pEM-GB1; this vector links a 6xHis-tag and the coding sequence of the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) to the N-terminus of the protein of interest 25,26 , thus enhancing solubility 36,37 . To examine in vivo activity of the His-GB1-SinR fusion protein, the respective coding sequence was amplified from pEM-GB1-sinR and cloned into the broad host range expression plasmid pSRKKm which was then conjugated into the S. meliloti sinRmutant (DsinR::synTer1) described below.
For production and purification of N-terminally His6-tagged ExpR, the expR gene was amplified via colony PCR from Sm2B3001 and cloned into the expression vector pET28a using NdeI and HindIII restriction sites.
To completely abolish sinR expression, a synthetic multiple terminator site (synTer1) 38 flanked by the sinR promoter (up to -7 bp from the sinR start codon) and the last 21 codons of sinR followed by the sinI promoter and most of the sinI coding sequence was cloned into pK18mobsacB; after integration of the resulting plasmid pK18mobsacB-DsinR::synTer1 into the S. meliloti genome, the resulting sinRstrains thus lacked sinR, but carried the sinR promoter and most of its UTR followed by the terminator sequence blocking potential read-through, the 3' end of sinR and the native sinI promoter and coding sequence.
To specifically reduce sinR expression levels, a mutated version of the sinR promoter in which the 5' half of the NurR binding site had been exchanged (GTTTATGAAATATTGCACTA to TGCCAAGCTTTATTGCACTA) according to 27 was cloned into pK18mobsacB either in front of sinR1-27-mScarlet-I (for the single molecule microscopy strain) or in front of sinR (for time-lapse microscopy and flow cytometry strains); due to presence of many restriction sites in the assembled fragment, only the vector was digested with EcoRI and BamHI, while the outer primers were designed with homology to the vector, and fusion of vector and insert was done by aqua cloning 39 . The mutated promoter was then stably integrated into the genomes of the respective strains by double homologous recombination.
For direct overexpression of sinR, the sinR coding sequence preceded by the T5 ribosome binding site (added upstream of sinR during amplification by means of a primer tail) was cloned into pSRKGm using NdeI/SacI restriction sites.
For overexpression of mScarlet-I-sinR, the two genes were amplified separately, sinR1-27-mScarlet-I as described for overexpression of sinR with preceding T5 ribosome binding site, and as described for mScarlet-I-sinR without stop codon, but including a linker. Both PCR fragments were then digested with BamHI, subsequently ligated, and the product was inserted into the NdeI/SacI-digested pSRKGm via aqua cloning.
The plasmid pK18mobsacB-expR ATGwas used to restore the AHL receptor in strains derived from S. meliloti Rm2011 as these strains carry an insertion sequence in the expR locus SMc03899-SMc03896 (expR::ISRm2011-1) 17 .

Supplementary Methods 2. Construction and characterization of the expR -pde0 strain.
The S. meliloti Rm2011 genome encodes 22 c-di-GMP-related proteins, 13 of which contain predicted phosphodiesterase (PDE) domains 5 . For the expR -pde0 strain, these 13 genes were deleted or, in case of SMc00074, replaced with a variant encoding an active site mutation via double homologous recombination using the suicide vector pK18mobsacB 21 . The table lists the S. meliloti Rm2011 genes containing PDE domains, the plasmids used for construction of the respective deletion strains, and either the primers used for plasmid construction or the plasmid source. pK18mobsacB-SMc00074-AAL contains a mutated gene encoding a variant of SMc00074 in which the 'EAL' motif essential for c-di-GMP degradation by PDE domains 41 is replaced for 'AAL' (E746A). All other plasmids contain 600-800 bp-long upstream and downstream flanking regions of the respective gene. Upstream flanking regions include the start codon plus ensuing 27 bp, downstream flanking regions include the last 27 bp plus ensuing stop codon. The primers given were used for amplification of the respective flanking regions from Rm2011 or, in case of SMc00074, for amplification of the gene variant from pABC-rgsPAAL 40 . Upper case letters in primer sequences indicate bases annealing to the PCR template, lower case letters indicate tails containing restriction sites. Deletions and the SMc00074-AAL mutation were introduced into Rm2011 (expR -) sequentially from top to bottom.

Quantification of c-di-GMP levels.
Quantification of intracellular c-di-GMP levels was carried out as described by Burhenne and Kaever 42 . Briefly, nucleotides were extracted from cell pellets of 5ml TY cultures using 40% (v/v) acetonitrile, 40% (v/v) methanol, and 20% (v/v) water. Samples were then dried and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Amounts of c-di-GMP detected were normalized to protein mass in the respective sample determined by Bradford assays.
Phenotype assays. Surface attachment was quantified with crystal violet staining as previously described 5 . In short, strains were grown to stationary phase in 30% MOPS-buffered medium (MOPS-buffered medium with nitrogen, carbon and phosphate sources reduced to 30%), diluted 1:10 in the same medium and subsequently grown in 96-well microtiter plates at 30 °C without shaking. After 2 days, cell densities were determined, the medium and unattached cells were removed, and the wells were washed with 200 µl water. Remaining (i.e., attached) cells were stained with 200 µl of aqueous 0.1% (m/v) crystal violet solution for 20 min at room temperature while gently shaking. The staining solution was then discarded, the wells washed twice with water, and the stain dissolved in 200 µl of 20% (v/v) acetone and 80% (v/v) ethanol for 20 minutes at room temperature. Staining of the solution was determined by measuring absorbance at 570 nm (A570) with an Infinite M Plex microplate reader (Tecan) and normalized to OD600.
Motility was quantified by spotting 2 µl of stationary phase cultures on a 1:5 diluted TY agar plate (final agar concentration 0.3% (m/v)). Plates were then incubated at 30 °C and imaged after 2 days. Spot diameters were measured with the Fiji/ImageJ image processing software.
Growth curves were determined by growing 3 biological replicates of each strain in the respective medium in a 96-well microtiter plate shaking at 200 rpm in an Infinite M Plex microplate reader (Tecan) set to 30 °C. Starter cultures had been grown in the same medium to stationary phase and diluted 1:100 for the experiment.